PROJECT DESCRIPTION Despite significant progress in deciphering the regulation of pluripotency in mouse models, there remain fun- damental gaps in our understanding of how human (h)ESCs maintain the pluripotent state Our long-term goal is to decipher the architecture of the regulatory network that allows for unrestricted hESC proliferation while preserving their potential to form the full repertoire of cell types found in the human body. To comprehensively identify genes involved in pluripotency maintenance we have conducted genome-wide shRNA screens in hESC and begun in-depth analyses of the most significant hits. We focused on BCOR, a component of the non-canonical Polycomb repressive complex 1.1 (PRC1.1) with a strong loss-of-pluripotency phenotype con- sistently observed in multiple hESC lines. BCOR depletion in hESCs led to a loss of repressive chromatin at key developmental loci and initiation of differentiation. We found that BCOR defines a novel subtype of the PRC1 complexes with distinct recruitment and repression mechanisms. This novel BCOR-PRC1.1 complex complements previously described KDM2B-PRC1.1 complex to efficiently silence differentiation programs in hESCs. Our central hypothesis, formulated based on the preliminary data and prior work in mouse and human ESCs, is that Polycomb repression of developmental regulators is established through combined action of the KDM2B-PRC1.1 and the BCOR-PRC1.1 complexes. KDM2B mediates PRC1.1 recruitment to accessible CpG islands while BCOR is responsible for the recruitment to dense chromatin, confers additional repressor function and facilitates the deposition of H3K27me3 at target loci. In Aim 1, we will define the mechanisms of targeting and repression by the non-canonical PRC1.1 complexes: (1A) Identify accessory factors and/or epigenetic marks required for BCOR-mediated PRC1.1 targeting; (1B) Determine how local chromatin structure affects the targeting of BCOR-PRC1.1 complexes. (1C) Define the repression mechanism mediated through the N- terminus of BCOR, and (1D) Delineate specific roles of the KDM2B-PRC1.1 and the BCOR-PRC1.1 complex- es in Polycomb domain assembly during the transition from nave to primed pluripotent state. Furthermore, in Aim 2, we will use our shRNA screen data to reconstruct the gene regulatory network that maintains primed pluripotency in humans. We will employ CRISPR-i technology to knock-down 130 transcription factors whose depletion leads to a loss of pluripotency. We will use single cell RNA-sequencing and our novel computational tools, MAGIC and PHATE, to infer regulatory gene modules from perturbations. We will use ChIP-Seq to de- fine genomic footprints of the key regulatory modules and integrate these data with hESC epigenetic map to discover novel mechanisms of transcriptional control. Our approach is innovative, because we utilize novel state-of-art technologies to obtain new insights into the regulatory complexity of hESCs. The proposed re- search is significant, because it is expected to expand understanding of cell fate regulation, define of key dif- ferences between hESCs and mESCs and guide development of new approaches for cellular reprogramming.